Reorganization of cellular membranes and transport of components from one lipid-bilayer bounded compartment to another underlie much of the internal structure of a cell. Clathrin is the principal molecular scaffold for a number of such processes -- most notably, receptor-mediated endocytosis of ligands such as transferrin, LDL, growth factors, and hormones. Viruses and other pathogens and pathogenic toxins usurp this pathway to enter cells. Direct observation of the biochemistry of clathrin-dependent membrane traffic in living cells is now possible, through a combination of genome editing and single-fluorophore sensitivity imaging. The combination, which amounts to in vivo, single-molecule biochemistry, resolves ambiguities about essential components and the time points at which they function, particularly when many of the steps have a stochastic rather than fully deterministic character. Our work in the previous grant period on the molecular mechanism of coated-pit initiation illustrates the value of single-fluorophore-sensitivity imaging approaches. In the research described in this proposal, we will analyze the molecular mechanisms of transitional checkpoints in coat assembly, develop and apply probes for the roles of specific phosphoinositide lipids in regulating clathrin- based membrane traffic, and extend our imaging biochemistry to the 3D context of multicellular assemblies and living tissues. We will capitalize on a transformative new imaging technology, lattice light- sheet microscopy (LLSM), which enables rapid, high-resolution, high-sensitivity 3D visualization of whole cells and multicellular specimens, including living tissues.